Identification of biological targets, typically proteins, physically bound by chemicals such as pharmaceutical compounds is an important step in the design, optimization and clinical evaluation of bioactive therapeutic agents such as drugs and their metabolic byproducts as well as in the elucidation of their mechanisms of action.
Drugs typically function by binding to one or more cellular proteins (hereafter referred to as the target(s)), thereby perturbing the bound protein's function, either activating (as in an agonist) or inactivating (as in an antagonist) the protein's biological activity by altering the target's enzymatic activity, protein folding, or the protein's interactions with other molecules, etc. Optimally effective drugs usually display high binding affinity and specificity for the target(s), ideally selectively interacting with only a single protein family member, isoform or variant. In contrast, sub-optimal drugs often exhibit more promiscuity (i.e. less specificity in binding), leading to unwanted and/or unanticipated biological perturbations of multiple “off-target” cellular proteins or pathways leading to undesirable, biological effects and/or toxicity.
Modern therapeutic development is critically dependent on a process of systematic screening and optimization of the medicinal chemical properties of chemical compounds to meet stringent criteria defining the target specificity, affinity, and stability. Therapeutic prioritization is often based on critical insights gathered from careful elucidation of structure-function relationships and related biophysical properties, in particular target specificity and binding affinity, to assess lead agents and prioritize the selection of pharmaceutically active compounds for advanced clinical trials. Compounds exhibiting preferential target affinity and selectivity are usually preferred over less specific chemically distinct compounds.
The metabolism of lead compounds and drugs by cells and tissues can produce metabolite isoforms that exhibit either enhanced or reduced binding affinity and/or specificity for a given protein target, which can affect the compounds effectiveness, bioavailability, mode of action and/or stability. Knowledge of the preferential binding of one drug metabolite variant to a target protein(s) relative to other lead compounds can provide insight into optimal medicinal chemistry (e.g. drug structure-function relationships). Conversely, knowledge of the protein partners physically bound by various drug metabolites in affected cells and tissues can provide insights into their respective mechanisms of action, their relative effectiveness, and their target specificity.
Modern drug discovery usually occurs via either (i) a target-driven compound screening approach, wherein libraries of small synthetic chemical molecules or natural compounds are tested for specific binding to a particular protein of interest (aka the “target”), or (ii) via phenotypic screens, wherein a panel of compounds is screened for a desirable biological effect(s) upon treatment of healthy or diseased cells, tissues or model organisms, followed by the isolation and identification of the protein(s) targeted (i.e. bound) by the drug. The former approach typically involves the complete or partial elucidation of cellular pathways implicated in physiological functions that are altered in the disease state as a means of identifying biochemically desirable candidate macromolecules targets for therapeutic development (e.g. target profiling by functional screening). This approach often generates ineffective or toxic lead compounds in clinical trials since they frequently suffer from unexpected “off-target” effects due to unwanted non-selective binding to other non-targeted proteins. A major limitation of the latter approach is the lack of knowledge of the mechanism of action (e.g. the target(s)) of a lead compound. In both scenarios, identification of the protein(s) bound to be the lead compounds and its various in vivo metabolites in as near a physiological context as possible (that is, in the presence of all the available binding partner proteins present in an organelle, cell, or tissue) is crucial to confirming or elucidating the exact mode of drug action. Such information is essential for the development and validation of safe therapeutic agents with well-defined clinical indications.
The process of identifying drug targets and elucidating their downstream cellular effects occupies considerable effort in basic and pharmacological research. Classically, the identification of protein targets for phenotype-perturbing small molecules has been accomplished biochemically using labeled or immobilized molecules. Yet the identification of the physical targets of a drug has often proven to be exceedingly difficult firstly because introduction of a functional group (i.e. chemical derivitization) that may be used for immobilization or detection of the drug-target complex often changes the bioactive properties of the drug itself. Secondly, different drugs show a very wide range of affinities for their primary targets and to a greater or lesser degree to a range of secondary “off-target” proteins. In combination, these factors make it almost impossible to design and evaluate experiments aimed at identifying primary drug targets through direct physical capture on a generic level.
Identification of the protein(s) directly involved in cellular pathways has been greatly facilitated by the recent developments in proteomics, especially with the introduction of effective methods for the rapid separation, purification and mass spectrometry analysis (identification and quantification) of cellular proteins and protein complexes in tissue, cell and organelle extracts. Once potential targets have been biochemically isolated and identified, they are typically expressed in a heterologous host system and purified in recombinant form suitable for in vitro assays. Drugs can then be screened against these targets in vitro. Screening for compounds that can bind to known potential targets in isolation can be achieved by techniques that are well known in the art and may include, for example, frontal chromatography, tracking of labeled chemicals for their binding to immobilized potential targets using techniques such as fluorescence microscopy and binding assays in general. Conversely, drugs can be modified chemically to add a tether suitable for affinity isolation and then incubated with cell extracts with the hopes of enriching and purifying the target protein(s).
While examples of successful identification of drug targets have been reported in the literature, these methods are often prone to failure and typically require sizeable logistical efforts that make them unsuitable or impractical for rapid pre-screening of large-numbers of lead compounds. These aforementioned methods of drug screening also usually require the characterization of cellular pathways, which can be tedious and may often only resolve parts of a particular pathway or network of interacting, redundant pathways. Furthermore, the above-described approaches do not permit rapid identification of cellular targets in cases where compounds are massively screened for their effect on cells. For example, compounds for which the cellular target(s) is unknown may be found to be effective in achieving a desired effect such as inhibiting cell growth, but subsequent identification of the target may be very difficult or impossible with classical approaches. Also, while a particular compound may target proteins in a particular pathway, other proteins may also be bound by the compound, which may affect or alter its pharmacological properties and cellular effects. Ignorance of the identity of these other protein targets may impede the development of improved drugs.
As mentioned above, several biophysical methods have been developed to elucidate the direct binding targets of small molecules and drug-like compounds. These include Frontal-Affinity Chromatography (FAC), Nuclear Magnetic Resonance (NMR), Capillary Electrophoresis (CE). While these methods vary in their sensitivity, throughput and potential applications, they are universally non-optimal for identifying the proteins binding partners of lead compounds, drugs, and their metabolites in the context of complex biological mixtures, such as tissue homogenates, cell and organelle extracts, and biological fluids like blood or cerebral spinal fluid. In particular, a major unaddressed need is an easily implemented and readily generalizable method for accurately and rapidly discovering and sensitively monitoring those proteins that are selectively bound by a drug and its metabolites in the context of the myriad of proteins present in an organelle, cell, tissue, organ or entire organism.
There is therefore a pressing need for better (i.e. effective and generic) screening methods to identify and confirm the cellular protein targets of drugs and lead therapeutic compounds in complex protein lysates that have been prepared from cultured cells and/or tissues of model organisms or human blood specimens in a physiologically relevant manner. For maximal flexibility, the method should allow for the elucidation of drug targets using protein extracts prepared either before or after in vitro or in vivo treatment with a bioactive and control agents of interest.